Field of the Invention
The present invention relates to a multi-layer test device, and more specifically, to analyzing a liquid sample for the concentration of fructosamine.
Background Information
Individuals suffering from diabetes mellitus have an abnormally high blood sugar level because the pancreas does not secrete sufficient amounts of the active hormone insulin into the bloodstream to regulate carbohydrate metabolism. If an abnormally high blood sugar level, known as a hyperglycemic condition, is allowed to continue for prolonged periods, the individual will suffer from the chronic complications of diabetes, including retinopathy, nephropathy, neuropathy and atherosclerosis.
It is known that proteins, such as those present in blood serum and other biological fluids, react with glucose, under non-enzymatic conditions, to produce glycated proteins, also known as fructosamines or ketoamines. The extent of the reaction is directly dependent upon the glucose concentration of the blood. Consequently, diabetics usually have an elevated fructosamine concentration compared to a healthy individual. Therefore, the concentration of glycated serum proteins has been used as an indicator of a hyperglycemic condition. To assist in the detection, monitoring and treatment of diabetes, blood sugar concentrations can be determined indirectly by analyzing for fructosamine. In particular, measurement of fructosamine levels is useful for monitoring diabetic control because fructosamine concentration reflects an average of serum glucose levels over a period of time, approximately a half month period.
Fructosamines are formed as follows. The blood proteins are glycated in vivo by a non-enzymatic reaction between glucose and available amino groups of blood proteins. The glucose binds to an amino group of the protein to form a Schiff base, i.e., an aldimine, that undergoes molecular rearrangement to form a stable ketoamine. In the art, such ketoamines are generically known as "fructosamines." The degree of protein glycation and fructosamine formation is directly proportional to blood glucose concentration.
Many of the existing methods for determining the degree of diabetic control are unreliable. For example, tests which directly measure plasma glucose have been found to be inadequate or even misleading because blood glucose levels fluctuate significantly throughout a given day, being influenced by diet, activity, and treatment.
Because of the shortcomings of direct glucose measurements, tests which are long term indicators of blood glucose levels were developed. One of the first such tests measured glycated hemoglobin. Because of the expense, requirement for trained technicians and lack of reproducibility associated with measuring glycated hemoglobin, more reliable tests of measuring for serum fructosamine developed. An assay for fructosamine overcomes some of the disadvantages of the assay for glycated hemoglobin because fructosamine measurements are technically simpler to perform and reflect a shorter time period of glycemic control (1/2 month) compared to glycated hemoglobin (2 to 3 months).
Baker, in U.S. Pat. Nos. 4,642,295 and 4,645,742, discloses methods for determining fructosamine levels. The disclosed methods however use a solution assay, which has several practical limitations, such as inconvenience associated with solution assays and the need for relatively large volumes of liquid sample. Such methods have been adapted to dry-phase chemistry, for example by Ismail, as disclosed in U.S. Pat. No. 4,956,301. However, Ismail's dry-phase assay is a test-strip device, which requires the use of a reaction accelerator compound and which is incapable of performing with certain reagents which are especially useful in fructosamine assays, namely Nitroblue tetrazolium chloride (NBT) and carbonate buffer.
Multi-layer test devices are generally known in the art. Many patents in the field, such as Goffe et al. U.S. Pat. No. 4,050,898, however, describe the need for a spreading layer. To overcome the use of spreading layers some multi-layer devices have an air gap between the first support member and the multi-layer analysis film, as shown in the published European Patent Application 0 388 782.
Sakamoto, in the published European Patent Application 0 473 189, describes a multi-layer analytical element for assaying fructosamine. The arrangement of the multi-layers, as well as the materials used, however, are different than the instant invention. As a result, Sakamoto's element requires the use of binders and is incapable of analyzing fructosamine levels in whole blood.
Therefore, a need still exists in the art for a simple dry-phase test device which involves only the minimal chemistry required for analyzing the concentration of fructosamine. And in particular, a need still exists for adopting this chemistry to a simple multi-layer test device which does not require a spreading layer or air gap and which can analyze the concentration of fructosamine in whole blood. The present invention satisfies these needs and provides related advantages as well.